Fluorescence in Cytometry
The power of cytometry arises from our ability to simultaneously mark and analyse several different cellular structures or properties with fluorescent dyes.
Immunofluorescence
In the immunofluorescence technique, fluorescent dye is carried to the cell by antibody molecules (usually monoclonal) that are specific for some cellular receptor. There are a wide range of fluorochromes that may be directly conjugated to antibody molecules or obtained already attached to forms of avidin, ready for subsequent binding to biotinilated antibody. Common examples are fluorescein, phycoerythrin (PE), allophycocyanine (APC), texas red, the cyanine dyes Cy3, Cy5 and Cy7 and Molecular Probes' Alexa dyes.
The Dyes That Bind
There are dyes that bind of their own accord specifically to intra-cellular sites. for example, Propidium Iodide binds to nucleic acid (DNA or RNA), Hoechst 33342 binds to AT-rich regions of double-stranded DNA, Rhodamine123 binds to mitochondria.
Keeping Track
Tracking dyes (e.g. CFSE, PKH26) bind tenaciously to cells, surviving for days in vivo, and even through several cell divisions, upon each of which the fluorochrome is divided between the daughter cells.
Intra-Cellular Chemistry
Some fluorochromes are even sensitive to the local chemistry (INDO-1 responds to intra-cellular Ca++ concentration, BCECF to intra-cellular pH).
Choosing Fluorochromes
Each fluorochrome has its own excitation spectrum (the range of illuminating light wavelengths that will cause it to fluoresce) and an emission spectrum (the spectrum of fluorescent light emitted). Since flow cytometers and confocal microscopes use laser light for illumination, the laser wavelength must be within the excitation spectrum. To use two dyes simultaneously, both of their excitation spectra must overlap the laser wavelength but their emission spectra must differ. More flexibility of choice is afforded where more than one laser is used.
The figure illustrates these ideas. It depicts the use of fluorescein and phycoerythrin (PE) excited by one laser at 488nm. Note that the laser is not at the peak excitation wavelength for PE but in a position to yield 56% maximal excitation. The fluorescein and PE emission spectra are sufficiently separate to allow optical filtering although they do overlap (a problem dealt with by fluorescence compensation). APC is excited by a second laser at 633nm, again not at peak excitation wavelength. This laser light wavelength must be far enough from the PE emission not to pass through the PE detector's optical filter.
A complex cytometric analysis can be designed according to these simple principles. For assistance in matching fluorochromes, laser wavelengths and optical filters, see FCMDesigner. For further discussion on the physical nature of fluorescence, see Exciting Fluorescence.